DNA Extraction

Transcription

DNA Extraction
DNA Extraction
Learning Objectives: Students learn about DNA, cell structure, and
basic chemical separations.
GRADE LEVEL
4–8
SCIENCE TOPICS
Solutions and Mixtures
Techniques
Organic and Biochemistry
PROCESS SKILLS
Describing and Defining
Explaining
Evaluating
GROUP SIZE
1–3
If available, goggles are
recommended for this activity.
SNEAK PEAK inside …
ACTIVITY
Students extract DNA from strawberries.
STUDENT SUPPLIES
see next page for more supplies
strawberries
sealing plastic bags
dish soap
salt
meat tenderizer
isopropyl alcohol, etc….
ADVANCE PREPARATION
see next page for more details
dilute soap
mix tenderizer and salt together, etc….
OPTIONAL EXTRAS
DEMONSTRATION
Modeling the Procedure (p. C - 22)
EXTENSIONS
Animal DNA (p. C - 29)
Other DNA Sources (p. C - 30)
TIME REQUIRED
Advance Preparation
Set Up
Activity
Clean Up
15 minutes
the day before
15 minutes
20 minutes
15 minutes
DNA Extraction
Grades 4–8
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2007, OMSI
SUPPLIES
Item
Amount Needed
strawberries
1 per group
sealing plastic bags (e.g., ZiplocTM)
1 per group
liquid dish soap
½ teaspoon per group
99% isopropyl alcohol (or lower, e.g., 70%
rubbing alcohol)
meat tenderizer
OR
papaya or pineapple juice
salt
tall, clear, narrow plastic cups (8 oz. or 12 oz.)
plastic spoon
pop-top squeeze bottles (e.g., water or sports drink)
freezer or bucket of ice
¼ cup per group
1 tablespoon per class
OR
¼ cup juice per class
1 tablespoon per class
2 per group
1 per group
1 per group
1 per class
For Extension or Demonstration supplies, see the corresponding section.
ADVANCE PREPARATION
Supplies Preparation
Strawberries:
‰ Purchase fresh or thawed, green tops on or off.
Isopropyl alcohol:
‰ 99% isopropyl alcohol is best.
‰ 90% or 70% (rubbing alcohol) will work also.
CAUTION: Isopropyl alcohol is flammable and poisonous. Keep away
from heat and open flames.
Soap solution:
‰ Dilute liquid dish soap with water: one part soap or detergent to
two parts water.
‰ If possible, leave the soap mixture in a refrigerated place until
immediately before use.
Either—meat tenderizer/salt mixture:
‰ Mix 1 tablespoon salt with 1 tablespoon powdered meat tenderizer
in a plastic cup.
DNA Extraction
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Or—papaya or pineapple juice:
‰ Use 1 cup of fresh, frozen (diluted as directed), or canned juice.
‰ Make sure juice contains raw, uncooked fruit juice.
Notes and Hints
‰ Keep the isopropyl alcohol very cold—use the freezer or ice bucket.
Give to students as close to the start of the activity as possible.
‰ Pass out the pinches of meat tenderizer/salt mixture yourself or put the
container at a central location for students. In the same way, you may
pass out the juice samples yourself or make the juice, with a ⅛
teaspoon measure, available to all the students.
‰ Tall, narrow cups work best to see the layers of alcohol and strawberry
mixture.
SETUP
For each group
‰ 1 sealing plastic bag
‰ ¼ cup cold isopropyl alcohol in a plastic cup
‰ 1 strawberry
‰ ¼ cup soap solution in pop-top squeeze bottle
‰ 1 teaspoon measure
‰ 1 clear plastic cup (6 oz. or larger, tall and narrow works best)
At a central location (or with the teacher)
sponges and towels for clean up
‰ a bucket of ice or access to a freezer
‰ Either—meat tenderizer/salt mixture (2 tbsp) in a plastic cup
Or—papaya or pineapple juice (1 cup) in a plastic cup, with
‰ ⅛ teaspoon measure
‰
INTRODUCING THE ACTIVITY
Let the students
speculate before
offering answers
to any questions.
The answers at
right are provided
for the teacher.
Choose
questions that
are appropriate
for your
classroom.
DNA Extraction
Grades 4–8
In this activity, students will explore some of these questions by looking at
the DNA from strawberries.
What do you think of when you hear the term “DNA”?
Most likely students have heard the term in daily life through newspapers,
radio, or television. It may come up in stories related to crime solving,
inheriting traits, treatments for disease, or identifying remains after an
accident.
C – 21
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What sorts of things have DNA?
All living things have DNA, including plants, animals, fungi, bacteria, etc.
Some viruses have DNA. Rocks, water, clouds, and other non-living things
do not have DNA.
What characteristics of people does DNA influence?
DNA is in charge of physical attributes like height, hair color, eye color,
etc. Dyed hair, colored contacts, tattoos, etc., are not determined by
DNA. Things like interests, hobbies, and weight can be influenced by DNA
but are also affected by how people grow up. Many scientists are still
researching exactly which characteristics of people are determined by
DNA and what are influenced by their environment.
Where do people/living things keep their DNA?
Many answers are possible, but students should know that a copy of DNA
is kept in each cell in a living organism. Cells are surrounded by protective
barriers (cell walls and membranes, see Explanation) that help organize
and keep the DNA safe.
If we want to get the DNA out of living things to a place where we can see
it, what do we need to do?
The DNA needs to be taken out of the cells. We need to get a source of
DNA (i.e., something living), break down its cell walls and membranes,
and then separate the DNA out from everything else in the cell. Students
do this in this experiment.
What do you think DNA looks like with the naked eye?
Students can draw pictures, or write a sentence or two. The double helix
structure might come up, or the “x” like structure of a chromosome.
Individual molecules of DNA are too small to see even with microscopes
(scientists use X-rays). On the other hand, thousands of strands of DNA all
clumped together are visible even to the naked eye.
TEACHER DEMONSTRATION
Modeling the Procedure
For younger students, it is a good idea to model the steps in this reaction,
especially layering the alcohol on top of the squished strawberry mixture
without letting them mix.
To layer the alcohol, tilt the strawberry cup and pour the alcohol SLOWLY
down the side of the cup so it doesn’t disturb the mixture. Alcohol and
water readily mix together, so take care to pour the alcohol slowly onto
the strawberry mixture.
DNA Extraction
Grades 4–8
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CLASSROOM ACTIVITY
Have students follow the Scientific Procedure on page C - 34, working in groups of 2–3. Below
are suggestions to help the teacher facilitate the activity.
NOTES
If available, goggles are
recommended for this activity.
This handout is
on p. C - 34.
Running Suggestions:
‰
‰
‰
‰
The colder the alcohol is, the more DNA it will extract. It can be
stored in the freezer without freezing solid.
During the activity, you might pour out ¼ cup of cold alcohol to
each group. This is safer than letting students walk around the room
with alcohol and also allows the alcohol to stay cold longer.
Make sure the plastic bag is closed tightly between steps.
The hardest step is layering the alcohol. Make sure students DO
NOT pour alcohol directly on top of the strawberry mixture at the
bottom of the cup. They should tilt the cup and pour the alcohol
SLOWLY so it flows down the side of the cup and floats on top of
the strawberry mess. If the alcohol and water layers mix, the DNA
will not precipitate out.
CAUTION: Students should never put lab supplies in their mouths. Even
if lab supplies are foods, they may be contaminated by dirty hands or
poisons in the lab.
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Ongoing Assessment
‰
‰
‰
Why do we need to squish the strawberry? (To free the DNA. Squishing
breaks cell walls and releases the contents of the cell.)
Does anyone have a guess what the soap and tenderizer is for? (The
soap dissolves the cell and nuclear membranes that protect the DNA.
The meat tenderizer and salt help control enzymes in the cells and
keep the DNA structurally intact.)
Now that we have added soap and tenderizer, we have the DNA out of
the cell. Why can’t we see it? (It’s still dissolved in the water from the cell.)
Safety and Disposal Information
‰
‰
‰
If available, goggles are recommended for this activity.
Caution students to never put lab supplies into their mouths. Even if they
are foods, they may be contaminated from other things in the lab.
Waste isopropyl alcohol should be kept away from all sparks and
flames. The small amounts used in this experiment may be poured
down the sink drain with lots of water. All other materials can be
thrown away as solid waste.
CAUTION: Isopropyl alcohol is flammable and poisonous.
CLASSROOM DISCUSSION
Ask for student
observations and
explanations. Let
the students
guide the
discussion and
present their
hypotheses
before discussing
explanations.
Choose questions
that are
appropriate for
your classroom.
DNA Extraction
Grades 4–8
What happened when you added the alcohol?
Snot, goo, white clumpy stuff, etc., came out of the strawberries and
floated on top.
If this is DNA from the strawberries, does it look like what you expected?
Students will have various answers—some may have expected to see
double helices or long ropy strands. In fact, people can’t see those
microscopic features. Instead, the thousands of strands of DNA from the
strawberries look like white goo. NOTE: Some of the white, clumpy material
may be coagulated protein mixed with the DNA.
Why would scientists want to extract DNA from cells?
This has a large number of answers, such as:
‰ To change the DNA. Creating genetically modified foods, for example,
could create crops that are resistant to certain pests/insects or that
grow larger or faster.
‰ To analyze the DNA. Some diseases are linked to differences in
people’s DNA, so studying DNA can help scientists find cures and
create vaccines.
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‰
To identify the DNA. Since everyone has different DNA, it can be used
to identify him or her. Forensic scientists, for instance, can identify
criminals from DNA samples left at crime scenes. In the same way,
biochemists can perform paternity tests or identify people from their
remains after accidents.
Could the DNA we extracted be used to identify or study the strawberries?
Unfortunately, DNA is quite fragile and the methods used in this
experiment tend to break it apart. Scientists therefore use special
chemicals and procedures to protect DNA. For example, they add
chemicals to control the acidity of the solution. Scientists use other
chemicals to cut DNA apart very precisely to look at one part at a time.
This process can take several days to several weeks—not like on TV!
What other things could we extract the DNA out of?
In principle, scientists can extract the DNA from any living thing, and many
spend their careers doing just that. (Students are encouraged to try the
other foods mentioned in the Extensions to this activity.)
Note that although any living thing has DNA that can be extracted, the
method in this activity is crude and quick and works well for only a few
living objects. DNA is fragile and it is difficult to extract a whole DNA
molecule (a chromosome) intact. Scientists use other methods to extract
DNA from different organisms.
EXPLANATION
This background information is for teachers. Modify and communicate to students as necessary.
In this activity, students extract the DNA from strawberries using only salt, soap, and
alcohol. Students may be very surprised at how much DNA is recovered in this simple
procedure. Strawberries are used in this activity because they have a lot of DNA that
comes out easily and is very visible.
BACKGROUND FOR ALL GRADES:
All living things are made of cells, and each cell contains a molecule called DNA. DNA
carries the genetic information that determines gender, physical appearance,
vulnerability toward disease, and many other parts of who we are. Every cell in our
body contains DNA. Even though cells can only be seen under a microscope, an
uncoiled piece of DNA can be 2.8 inches (7.2 centimeters) long!
DNA Extraction
Grades 4–8
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2007, OMSI
Getting at the DNA
Plant and animal cells store their DNA in the nucleus, a small compartment inside each
cell that is surrounded and protected by the nuclear membrane (see Figure 1 below). A
membrane is a flexible, thin film that only water and a few special chemicals can pass
through. The cell membrane surrounds and protects the entire cell. Adding detergent or
soap breaks the nuclear and cell membranes, releasing the DNA.
Plant cells, but not animal cells, also have a rigid cell wall around the outside of the cell.
The students must smash the strawberry in the bag to break the cell walls and expose
the membranes inside to the soap or detergent.
Once the DNA has been released, the meat tenderizer (or pineapple or papaya juice)
helps untangle and unfold the DNA from the other parts of the cell.
Protecting the DNA
Breaking the nuclear membrane exposes the DNA to reactive and possibly dangerous
chemicals in the cell. To slow down these chemical reactions, which might damage or
chop up the DNA, students use cold ingredients and add cold alcohol.
Separating Out the DNA
Although it is free to float around, the DNA is still dissolved in water after it escapes the
membranes and cell wall. Students add alcohol, which floats on top of the water, to lift
the DNA out of the water and to separate it from the rest of the cell debris. Since the
DNA does not dissolve in alcohol, it precipitates (turns to a solid) in the alcohol layer. It is
visible as a kind of white goo.
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Grades 4–8
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2007, OMSI
EXTRA BACKGROUND FOR 6–8 GRADES:
Lipid Structure of Cell Walls and Membranes
In this activity, students add soap to lyse (break open) the cell and nuclear membranes
and release the DNA. Soap dissolves these membranes because they are basically
layers of oil that surround the cell.
In other words, dish soap destroys cell membranes in the same way that it cleans oil off
dishes and pans. Cell membranes and oil are both made of molecules called lipids.
Lipids are large molecules that have two parts: a small, compact hydrophilic head and
a long, dangling hydrophobic tail. (Hydrophilic means attracted to water, and
hydrophobic means repelled by water.) In lipids, most of the molecule (the tail) is
repelled by water, while the tiny head is attracted to water.
This head and tail structure of lipids allows them to arrange as large, two-layer sheets
when they are in water. The sheet structure allows the hydrophilic, water-loving heads
to be exposed to the water, while the hydrophobic, water-hating tails can be tucked
into the interior of the sheet. The cell and nuclear membranes are such sheets of lipids,
each with the water-attracting heads on the outside and the water-repellant tails on
the inside.
Lipids form two-layer sheets (i.e.,
membranes) with the water loving
heads pointing toward the water, and
the water hating tails pointing inside.
Figure 2. Lipids form two-layer
sheets as cell membranes. The
water loving heads point
toward the water, and the
water hating tails point away
from the water.
Soap and Cell Membranes
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Soap molecules are also lipids, just like the molecules in the membranes, but with one
key difference: their hydrophilic, water-loving head is more able to dissolve in water—
yet they still have a bit of a hydrophobic, water-hating tail. This means that they are still
“lipid enough” to mix with the lipids in the cell membrane, but also “water enough” to
mix with the water around the membrane. Because they can mix with lipids and water,
soaps and detergents move into the membranes and break them apart into greasy,
soapy bubbles (see the picture below).
Figure 3. Lipids have two parts: a
hydrophobic (water hating) tail and a
hydrophilic (water loving) head.
Soaps are lipids and so are the
membranes in cells.
Soap lipids are different from membrane
lipids because their hydrophilic “heads”
are more “water loving” and more able
to mix with water.
The membrane lipids and the soap lipids form little droplets with the membrane
molecules broken up between soap molecules.
Protecting the DNA
To prevent viruses and bacteria from invading the cell with their DNA, cells have
enzymes (called DNase enzymes) that chop up any DNA floating around outside the
nucleus. Once the strawberry DNA is out of the nucleus, it becomes vulnerable to these
enzymes. The students use cold ingredients to protect the DNA from DNases. Since
reactions are slower at lower temperatures, using cold alcohol and other cold
ingredients slows down the DNase reactions that break down DNA.
Meat tenderizer also helps to protect the DNA. Meat tenderizer contains another two
enzymes, bromelain and papain, which are extracted from pineapples and papayas,
respectively. (Pineapple and papaya juice perform the same function in this
experiment as meat tenderizer.) When DNA is packaged in the nucleus, it is wound
tightly around proteins. Bromelain and papain enzymes break down these proteins and
release the DNA with minimal breakage.
DNA Extraction
Grades 4–8
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Separating the DNA
DNA is soluble in water, but not in salty water. All the other cell contents are soluble in
water and salt water. This difference in solubility allows students to separate DNA from
the rest of the cell. Students add salt to the mixture to force the DNA out of the solution
of water, strawberries, and soap.
For more information on this process of separating substances according to their
solubility, see the activity Salting Out.
DNA is not soluble in alcohol. When the DNA leaves the salty water mixture, it comes
into contact with the alcohol and cannot dissolve. When DNA comes in contact with
alcohol, it uncoils and precipitates (turns into a solid that forms from a liquid solution). All
the other cell contents stay in the salty water solution.
Strawberries Have a Lot of DNA
Strawberries are octoploid, which means they have eight copies of their genetic
material. (As a comparison, humans are diploid, since we have two copies of our
genetic material.) This large volume of DNA ensures that even if the procedure is not
very accurate, or if mistakes are made, enough DNA can still be extracted to be visible.
EXTENSIONS
Extension A: Animal DNA
Extract DNA from animal cells and compare it to the extraction from plant cells.
Extra Supplies
‰ source of animal DNA (calf liver or thymus work well)—about 1 tablespoon per group
‰ blender
Extra Instructions
‰ Use the same procedure as the regular activity, but puree the liver or thymus in a
blender just before class. (These items don’t squish in bags very well.) Students
should receive a strawberry-size amount of the animal source.
‰ After the procedure, compare the animal DNA to the plant DNA from strawberries.
How is it the same and different?
CAUTION: Advise students using these raw meat products to wash their hands
thoroughly with soap and warm water after handling them.
Explanation
Animal DNA may separate more cleanly than plant DNA. Plant cells have cell walls,
which can separate out in the solution and be visible as a third layer. Because
animal cells lack cell walls, this layer doesn’t appear.
DNA Extraction
Grades 4–8
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2007, OMSI
Extension B: Other DNA Sources
Students can try to extract DNA from other living things using the same procedure
as for strawberries.
Extra Supplies
‰ other sources of DNA (some good ones are: wheat germ, spinach, peas, onions,
broccoli, bananas, and zucchini)—use about 1 tablespoon of each per student
group. Frozen foods work fine.
‰ blender—1 per class
Extra Instructions
Follow the same procedure as before but with the following changes:
‰ Firm sources (wheat germ, broccoli, onions, or zucchini) should be pureed in a
blender or food processor immediately before use.
‰ If possible grind the wheat germ in a coffee grinder first. Then soak in warm
water 5–10 minutes before blending.
CROSS-CURRICULAR CONNECTIONS
SOCIAL STUDIES
Genetically Modified Organisms
Find articles about genetically modified organisms (GMOs). What
products contain GMOs? What are the advantages and disadvantages
to GMOs? Since this is a controversial topic, stress to students the
importance of getting scientific summaries and not just information from
advocacy groups.
Discovery of DNA’s structure
Scientists knew information was somehow stored and passed on
between generations but didn’t know details. Use one of the books listed
in the Resources section to learn about how DNA’s structure was
discovered.
BIOLOGY
Cell Biology
Discuss the parts of the cell and their functions.
Discuss the differences between animal and plant cells.
DNA Extraction
Grades 4–8
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RESOURCES
Web – http://learn.genetics.utah.edu/units/activities/extraction/
This site has excellent pictures of the extraction process with diagrams of how detergent breaks apart fats
and cell membranes, as well as where in the cell the DNA is stored. The Frequently Asked Questions section
has additional information.
Web – http://jchemed.chem.wisc.edu/HS/classAct/ClassActsList.html
The Journal of Chemical Education publishes online lesson plans for high school students on multiple topics.
Under the topic, “Biochemistry,” the lesson “Liver and Onions” uses a more complicated procedure to
extract DNA.
Balkwill, Frances R., DNA is Here to Stay
Reading level: kindergarten to 4th grade
This gives a simple explanation of what DNA is and what it does in the body. This author also writes a series
called “Enjoy Your Cells.” This book is scientifically accurate and good for all ages.
Walker, Richard, Genes and DNA
Reading level: 4th to 8th grade
This book explores modern genetics, from an investigation of genes and their function to forensics, gene
therapy, and cloning.
Claybourne, Anna et al., Usborne Internet Linked Introduction to Genes and DNA
Reading level: 4th to 8th grade
Beautiful artwork takes you deep inside a cell. This book also explains genetically modified foods, the
Human Genome Project, gene therapy, designer babies, and DNA testing.
Watson, James D., The Double Helix: A Personal Account of the Discovery of the
Structure of DNA
Reading level: 10th to 12th grade
This is an autobiographical account from one of the discoverers of the structure of DNA. Tends to be a bit
dramatic.
Maddox, Brenda, Rosalind Franklin: The Dark Lady of DNA
Reading level: 10th to 12th grade
Rosalind Franklin conducted crucial research that led to the discovery of the double helical structure of
DNA. Because of her unpublished data and photographs, Francis Crick and James Watson were able to
publish their work on the structure of DNA.
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Grades 4–8
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VOCABULARY
bacteria:
very small organisms each made of just one cell
bromelain:
enzyme from pineapples that cuts up proteins
cell membrane:
a thin barrier that surrounds the contents of plant and animal
cells; provides structure and organization to the cell and controls
the passage of water and other chemicals both into and out of
the cell
cell wall:
the rigid outermost barrier that surrounds the cell membrane;
found in all plants and some algae, bacteria, and fungi; absent
from all animal cells
diploid:
having two copies of genetic material
DNA:
Deoxy-riboNucleic Acid; a long molecule found in the nucleus of
a cell and shaped like a double helix; associated with the
transmission of genetic information
DNase:
an enzyme that breaks down DNA; “-ase” stands for enzyme
enzyme:
complex protein produced by cells; acts to speed up a specific
biochemical reaction
hydrophilic:
attracted to water; easily absorbs water and dissolves in water
hydrophobic:
repelled by water; unable to absorb water or dissolve in water
lipids:
group of organic molecules that includes oils, fats, and waxes;
lipids, carbohydrates, and proteins make up the main structures
of cells
lyse:
to break open or split
membrane:
a thin film that forms a barrier
nuclear membrane: similar to the cell membrane; surrounds the contents of the
nucleus, separating it from the rest of the cell
nucleus:
a compartment inside the cell that contains the cell’s genetic
information
octoploid:
having eight copies of genetic material
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papain:
enzyme from papayas that cuts up proteins
precipitate:
to come out of a liquid solution as a solid
proteins:
a large, complex biological molecule found throughout the
body; hormones, enzymes, and antibodies are all proteins, as are
many of the structural parts of the body
soluble:
able to dissolve in a particular substance
virus:
a simple, small infectious agent made from genetic material with
a thin protein coat; cannot live without entering a cell so is not
considered living
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Grades 4–8
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DNA Extraction
SCIENTIFIC PROCEDURE
1.
Place a strawberry in a plastic bag. Close the bag tightly. Smash and squish the
strawberry until it forms a smooth paste.
•
What does the strawberry look like?
2.
Open the bag.
3.
Add one spoonful of the soap mixture into the bag.
4.
Add a pinch of the mixture of meat tenderizer and salt into the bag.
5.
Close the bag tightly and squish the contents until completely mixed.
•
How has the strawberry mixture changed?
6.
Open the bag and pour the contents into a clear plastic cup.
7.
SLOWLY pour alcohol so it runs down the side of
the cup and gently floats on top of the
strawberry mixture.
•
8.
Put the cup down and wait 30–60 seconds.
•
9.
You can tilt the strawberry cup to make this
easier. Continue to carefully pour until about 1”
of alcohol is on top of the strawberry mixture.
What is happening to the strawberry mixture?
Clean up your area.
•
Follow your teacher’s instructions.
SUPPLY WORKSHEET
This worksheet is available online at www.omsi.edu/k8chemistry.
DNA Extraction
Recommended group size: 2–3
Number of Students:
Supplies
Number of Groups:
Amount Needed
strawberries
1 per group
sealing plastic bags (e.g., ZiplocTM)
1 per group
liquid dish soap
½ teaspoon per group
99% isopropyl alcohol (or lower,
e.g., 70% rubbing alcohol)
meat tenderizer
OR
papaya or pineapple juice
Supplies
on Hand
Supplies
Needed
¼ cup per group
1 tablespoon per class
OR
¼ cup juice per class
salt
1 tablespoon per class
tall, clear, narrow plastic cups
(8 oz. or 12 oz.)
2 per group
teaspoon measure
1 per group
pop-top squeeze bottles
(e.g., water or sports drink)
1 per group
freezer or bucket of ice
1 per class
Extension A
source of animal DNA (calf liver or
thymus work well)
½ cup per class
blender
1 per class
Extension B
other sources of DNA (wheat germ,
spinach, peas, onions, broccoli,
bananas, zucchini)
blender
1–2 cups per class
1 per class
Teacher Demonstration
no extra supplies needed
DNA Extraction
Grades 4–8
C - 35
Chemistry in the K–8 Classroom
© 2007, OMSI
DNA Extraction
Grades 4–8
C - 36
Chemistry in the K–8 Classroom
© 2007, OMSI